Several techniques for extraction of antigens from micro organisms are well known to those skilled in the art. Cellular antigens such as carbohydrates, proteins, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, carbohydrates complexed with proteins, lipids, and others have been extracted by a variety of chemical or mechanical means. Such extraction techniques include boiling, autoclaving, detergent extraction, acid extraction, digestion with hot formamide, nitrous acid extraction, sonication or other mechanical disruption, phenol/chloroform extraction, or digestion by enzymes. If an antigen is to be used in an immunological assay it is important that the extraction protocol preserve the binding characteristics of that antigen.
Nitrous acid is a chemical extraction technique (Manual of Clinical Microbiology, Third Edition, Edwin H. Lennette, Albert Balows, William J. Havsler, Jr., and Joseph P. Traunt, Editors, American Society for Microbiology, Washington, D.C. (1980)). The method, traditionally, involves mixing two liquid reagents, an acid and a nitrite salt solution. This mixture generates a nitrous acid intermediate in solution. This nitrous solution is generally neutralized prior to subsequent steps. Assay kits based on this method include three separate reagent bottles designed to deliver a predetermined volume, i.e. , a number of drops, of each reagent to a sample tube.
Variations of this technique have been described. For example, Sand et al. (patent application, WO93/15217), describes an extraction of Group A or B Streptococci with two dried and one liquid reagent. The sodium nitrite and Tris base awe dried onto separate filter pads and an excess volume of acetic acid is added to reconstitute first the NaNO.sub.2, and then (after an extraction period) the Tris base which neutralizes the solution.
U.S. Pat. No. 4,851,337, describes another modification of the nitrous acid extraction protocol in which one vessel contains a polymeric acid and one contains a nitrite solution. While not reducing the number of reagents, this method provides pre-measured solutions that can be quickly mixed to allow antigen extraction. The end-user is not required to measure a specified number of reagent drops to the sample tube. In this method, the nitrite solution is transferred to a tube containing dried polymeric acid, mixed, and a sample swab added. After an incubation period, the solution is neutralized by the addition of a reagent from another tube.
U.S. Pat. No. 4,673,639, describes another modification of the nitrous acid extraction protocol, using microtubes containing two dried reagents in discrete zones of the tube. Reagents are reconstituted and activated by the addition of water and the sample containing swab. Reagents are held within the tube by inert binders or carriers such as dextran, polyacrylamide, polyacrylic acid, polyvinyl alcohol, PEG, PEO, PVP, guar gum, caboxymethylcellulose, hydroxyethyl cellulose, methyl cellulose, algin, carrageenan, and xanthan gum.
Extraction of antigens is an important step in the identification of micro organisms that are related to various disease states, such as Group A Streptococcus and Group B Streptococcus (GBS). Once the organism's antigens are extracted, they may be used for isolation and purification of a specific material for subsequent production of antibodies or vaccines or a variety of other uses. The extracted antigens which are specific to a given organism, may be used for the identification of the that organism from a test sample. The test sample may be any clinically relevant sample, such as serum, blood, urine, plasma, sputum, semen, throat swabs, vaginal swabs, and various secretions or other fluids. Of particular interest is the use of an immunoassay technique for the identification of a specific antigen for the diagnosis of a specific infection.
There are a variety of immunoassay techniques available. All rely on the ability of an antibody and its corresponding antigen to specifically interact. Latex agglutination relies on particle aggregation, colored or white, for the detection of a specific interaction. Antibodies may be labeled with radioactive, enzymatic, metallic particles, fluorescent, chemilluminescent, or a variety of other labels to produce an interpretable signal. In a classic form of immunoassay, an enzyme labeled antibody is used to convert a colorless substrate to a colored product which may be measured spectrophotometrically. Another immunoassay technique involves optical immunoassays. These techniques rely on the unique interactions of thin films with light to measure a mass change on the thin films and are described in U.S. Ser. No. 08/075,952, filed Jun. 11, 1993, entitled "Devices and Methods For Detection" is incorporated by reference herein, including drawings.